The health and integrity of photoreceptor cells depend critically on the composition and volume of their microenvironment. Ionic homeostasis in the subretinal space is in large part accomplished by the transport and barrier functions of the retinal pigment epithelium (RPE), a monolayer of cells juxtaposed between the photoreceptor outer segments and the choroidal blood supply. Transport of molecules between the photoreceptors and choroid involves the coordinated activity of a diverse group of pumps, cotransporters, exchangers, and channels residing in the apical and basolateral membranes of the RPE. With changes in retinal activity, chemical signals released by retinal cells diffuse to the RPE where transport is adjusted to compensate for alterations in the photoreceptor microenvironment. At the transport protein level, this modulation may involve covalent and non-covalent binding of organic and inorganic molecules. Disruption of these transport processes or their regulation may cause adverse changes in the photoreceptor microenvironment leading to retinal disease. These same transport pathways are responsible for maintaining the intracellular composition in the RPE cell, which if disturbed could adversely affect other key RPE functions such as vitamin A transport and metabolism and phagocytosis of photoreceptor outer segments. Our overall goal to understand the mechanisms by which ion channels participate in the regulation of the ionic composition of the fluid in both the subretinal space and RPE cytoplasm. The specific aims are: (1) To determine how potassium channels are regulated by intracellular calcium, ATP, and other factors; (2) To identify the specific types of chloride channels present in mammalian RPE and how they are regulated; and (3) To identify the conductive pathways underlying sodium and calcium entry into the RPE cell. These aims will be pursued using patch-clamp technology to record macroscopic and single-channel currents in freshly isolated human and bovine RPE cells. Another aim (4) is to confirm in intact tissue the activity and membrane location of these ion channels using intracellular microelectrodes in the isolated RPE-choroid preparation. These studies will result in a better understanding of how individual transport proteins are regulated to maintain normal transport function in the RPE and a healthy photoreceptor microenvironment.